Circadian rhythmicity is the overt manifestation of an innate timekeeping mechanism i.e., a "circadian clock." The clinical and practical importance of the human circadian timing system has now been recognized, and disordered clock function may underlie the symptoms of a number of sleep disorders and neuropsychiatric illnesses. The overall objective of this research program is to better understand the nature of the endogenous circadian pacemaker in the suprachiasmatic nuclei (SCN) in the hypothalamus of mammals. Two new research strategies are introduced in this competing continuation application. The first set of proposed experiments will study the circadian pacemaker without the interference of drugs or lesions by investigating natural genetic variations in pacemaker properties. In preliminary data, two inbred strains of mice (BALB/cByJ and C57BL/6J) were found that exhibited a large interstrain difference in the circadian period of their locomotor rhythms and a single strain (SEC/1ReJ) was found with a locomotor rhythm that exhibited an anomalous phase relationship to the external light-dark cycle. In proposed experiment, first, the physiological properties of the pacemakers in these strains will be documented by characterizing drinking and temperature rhythmicity, testing for possible aftereffects of prior environmental illumination, assessing the influence of testosterone, and constructing pacemaker "phase- response curves." Second, the structure and function of the SCN in these strains will be compared by delineating SCN architecture, mapping the patterns of SCN neurotransmitters and the density of SCN synaptic contacts, and determining SCN glucose utilization. Third, these interstrain differences will be used as experimental tools in neural transplantation and classical genetic experiments. The second set of proposed experiments will study the circadian pacemaker in a more controlled setting by investigating the SCN as an in vitro preparation. In preliminary data, the SCN was cultivated as a hypothalamic slice for weeks in vitro using the roller-tube method. In proposed experiments, first, the structure and metabolism of the cultured slice will be characterized by immunocytochemically labeling SCN neurons and devising a quantitative 14C-labeled deoxyglucose method for in vitro use. Second, circadian rhythmicity of the cultured slice will be tested by using assays of 22Na+ uptake, glucose utilization, and arginine vasopressin release.